The mechanisms that regulate the intake of food are vital for survival and health. While too little food consumption can lead to death, an equal or greater problem in 21st century America is the over-consumption of calorie-dense and inadequate consumption of vitamin-rich foods, resulting in obesity and poor nutrition. CB1 cannabinoid receptors (CB1R) in the brain play a vital role in the regulation of food intake, while activation of CB1R in peripheral tissues results in increased fat and glycogen storage; reduced glucose utilization by muscles; and reduced thermogenesis in brown adipose. Endocannabinoids are present in the human circulation and their concentrations are regulated by metabolic state and exhibit a circadian pattern. These previous findings lead us to the overall hypotheses that endocannabinoids in the circulation convey information about the need for additional caloric intake and that sustained, high endocannabinoid concentrations contribute to overeating. The objectives of this proposal are to establish and characterize an assay system in which endocannabinoid concentrations in the circulation of awake, freely behaving rats are determined. We will examine changes in endocannabinoid concentrations across the daily cycle; and following acute food restriction and stress, as these have all been shown to impact circulating endocannabinoid concentrations in humans. We will carry out three specific aims. The first aim is to optimize the endocannabinoid measurement in rat plasma; determining the limits of detection and quantification, and robustness. The second aim is to apply the assay to measure the circadian changes in circulating endocannabinoid concentrations in relationship to other metabolic signaling molecules; and to examine the effects of a brief period of food deprivation. The third aim will examine the time course of the effects of fear-evoking odor presentation on circulating endocannabinoids. Successful completion of these studies will provide the basis for future studies, including exploration of the mechanisms by which changes in energy status affect endocannabinoids in the circulation; the tissue source of the endocannabinoids in the circulation; and, most importantly, whether the endocannabinoids entering the brain from the circulation convey information about metabolic status to brain reward circuit activity. These studies are highly translatable to humans because endocannabinoid concentrations are easily measured in human blood. Indeed, the innovation of this project lies in its beside to bench translation of human observations to an animal model that is amenable to mechanistic studies. The studies in this project will increase our understanding of several processes important in the regulation of circulating endocannabinoid concentrations and will set the stage for future human and animal studies of their role in eating disorders, drug addiction and mood disorders.